Speculation has surrounded the future of both Duffen and manager Phil Brown in recent days, and the former has now officially vacated his post.
A club statement confirmed: "The club has received a letter of resignation from Paul Duffen, executive chairman of Hull City AFC and Superstadium Management Company Ltd, which has been accepted and is effective immediately."
Duffen took over at the KC Stadium from Adam Pearson in 2007 and oversaw the club's rise to the top flight for the first time in their history, which was achieved via victory in the 2008 Coca-Cola Championship play-off final.
Pearson on Wednesday resigned as chairman of football as Coca-Cola Championship club Derby, sparking suggestions he is set to return to East Yorkshire.
Hull avoided relegation on the final day of last season but chairman Duffen and manager Brown both came under pressure following their poor start to this campaign, which has seen them take just eight points from their opening 10 games.
The statement continued: "The club wishes to express its gratitude to Paul for his outstanding contribution as chairman during a period of unprecedented success for Hull City AFC."
The club will reveal more on their future plans next week, with the statement concluding: "The recruitment process has commenced in respect of the appointment of a new executive chairman and a further statement in relation to this will be issued by the club on Monday 2nd November."